The involvement of interdigitating (antigen-presenting) cells in the pathogenesis of rheumatoid arthritis.

Macrophage like cells expressing high concentrations of HLA-DR antigen have been identified in situ within the synovium of patients with rheumatoid arthritis. The characteristics of these cells have been determined using immunohistological analysis and combined cytochemical techniques. It was found that the majority (greater than 80%) of these cells were interspersed within the perivascular lymphocytic infiltrates occurring in the synovium. These cells did not stain with antisera against surface immunoglobulin or any Mc Abs to T lymphocyte markers. Further combined staining demonstrated that the HLA-DR + ve cells did stain with an anti-monocyte monoclonal (FMC-17), but could not be stained with a Mc Ab against C3b receptors. The interfacing of cytochemical reactions for acid phosphatase (ACP) and adenosine triphosphatase (ATPase) with immunofluorescence staining for HLA-DR demonstrated that these cells were ACP - ve ATPase + ve. This analysis led to the conclusion that the HLA-DR + ve cells found in abundance in the rheumatoid synovium expressed identical characteristics to the interdigitating cells of the normal lymph node paracortex. The possible significance of the presence of large numbers of such antigen presenting cells in the rheumatoid synovium is discussed.     

The use of laboratory intervention to stem the flow of fresh-frozen plasma

Four methods of laboratory intervention were tested by the hospital blood bank in an effort to modify the use of fresh-frozen plasma (FFP). Over a one-year period, a utilization audit was serially initiated with feedback to physicians, a recurrent educational program was introduced for housestaff delineating guidelines for FFP use, a form was introduced requiring justification for FFP orders, and a policy was established requiring pathologist approval of FFP in patients with normal or no coagulation studies. Overall, in comparing the period following all forms of intervention (February 1986-October 1986) to the baseline period prior to any form of intervention (July 1984-March 1985), FFP use dropped 52% in the face of a 17% increase in red blood cell use. It was concluded that blood bankers can dramatically alter the use of this product using established methods for modifying physician ordering behavior.     

Comparative virulence of blood and stool isolates of Shigella sonnei.

Shigellemia is rare in developed countries and might result from the emergence of unusually virulent strains. We compared systemic invasiveness markers of isolates from the blood of 3 temporally clustered patients with Shigella sonnei bacteremia in Boston with those of 11 unrelated contemporaneous strains from stools of people in New England. We found no difference between the two groups in O-chain length by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, mouse 50% lethal dose, in vivo response to iron, and susceptibility to serum, which varied from moderately susceptible to ultrasusceptible. Mean intraperitoneal 50% lethal doses of smooth form I colonies for mice were equally low (10(5.8) CFU) in both groups, and the 50% lethal doses were lowered equally further in the two groups by predosing with iron to levels useful in mouse model sepsis studies. S. sonnei bacteremia may reflect compromised host defenses, not bacterial virulence.     

Confirmation that the Type I collagen gene on chromosome 17 is COL1A1 (α1(I)), using a human genomic probe

A cloned 15 kb genomic fragment from the human α1 (I) collagen gene (COL1A1) has been used as a probe on restriction digests of DNA from human-mouse somatic cell hybrids. Positive results on hybrids containing chromosome 17 as their only karyotypically visible human material confirm the assignment of this gene to chromosome 17. Hybrids which contain fragments of chromosome 17 are used to confirm the localization to 17q21-qter.     

The lipid raft microdomain-associated protein reggie-1/flotillin-2 is expressed in human B cells and localized at the plasma membrane and centrosome in PBMCs

Reggie-1/flotillin-2 is a plasma membrane-associated cytoplasmic protein, which defines non-caveolar raft microdomains. Reggie-1/flotillin-2 is enriched in detergent insoluble (TX100) membrane fractions (DIG), co-localizes with activated GPI-linked proteins and the fyn-kinase in neurons and T cells, and thus apparently participates in the assembly of protein complexes essential for signal transduction. In T cells activated by crosslinking the GPI-linked protein Thy-1 or by crosslinking the ganglioside GM1, reggie-1/flotillin-2 co-localizes with the T cell receptor. To determine whether reggie-1/flotillin-2 is also expressed in B cells, primary B cells from human blood and cell lines representing the developmental stages of pro, pre, mature and plasma B cells were analyzed by Western blotting, RT-PCR and immunofluorescence. Here, we show that reggie-1/flotillin-2 is expressed throughout B cell development, as well as in primary B cells, purified by cell sorting. On non-activated mature B cell Raji cell line we found reggie-1/flotillin-2 are exclusively in the detergent (TX100) insoluble membrane fractions that are staining positive for the raft marker GM1. Immunofluorescence microscopy showed that reggie-1/flotillin-2 is localized at the plasma membrane and marks intracellular spots in PBMCs. Confocal co-localization studies showed that reggie-1/flotillin-2 is associated with the plasma membrane, and the centrosomes (microtubule organizing centers) in these PBMCs. Comparison of reggie-1/flotillin-2 cDNA sequences with the genomic sequence database allowed us to determine the exon/intron structures in mouse and human. The gene organizations are highly conserved suggesting an important function of reggie-1/flotillin-2. Since reggie/flotillin proteins co-cluster with the T cell receptor and fyn kinases upon T cell stimulation, our findings of reggie-1/flotillin-2 in B cells suggest a similar role in B cell function.     

Gene conversion,unequal crossing-over and mispairing at a non-tandem duplication during meiosis of Saccharomyces cerevisiae

Summary We have developed a novel system to examine conversion, exchange and mispairing involving a nontandem duplication of the ade8 locus in yeast by monitoring the segregation of heterozygous markers between the duplicated sequence. Plasmid Yrp 17 carries the yeast selectable markers URA3 ⁺ and TRP1 ⁺. Yrpl7 derivatives with a 4 kb insert carrying ade8-18 were used to clone the mutations trpl-1 and ura3-1 by gap repair. Integrants of the resulting plasmids at the Ade8 locus were crossed to yield diploid hybrids with a non-tandem duplication of Ade8 and heterozygosity for the plasmid markers between the duplicated sequences. 1192 complete, unselected asci were analyzed and 270 exhibiting recombination of the markers contributed by the plasmid were analyzed by Southern transfers to detect changes in plasmid sequences. Twenty-seven tetrads had unequal homologous exchanges and five had unequal sister-chromatid exchanges. Seven tetrads carry an additional copy of the integrated plasmid and ten are missing one. We propose that these two classes represent conversions of the entire 11 kb plasmid, which occur after misalignment and formation of an unpaired loop. Mispairing is a frequent event, and occurs in approximately fifty percent of all meioses. The system described provides a means to determine the meiotic rules of conversion, exchange and pairing for duplicated DNA sequences.     

Plasmids of Ewingella americana: supplementary epidemiologic markers in an outbreak of pseudobacteremia.

During an outbreak of pseudobacteremia in a children's hospital, Ewingella americana was found in blood cultures from 20 patients. E. americana was inoculated into blood culture bottles at the time of specimen collection due to cross contamination from nonsterile, citrated blood collection tubes used for coagulation studies. Antimicrobial susceptibility testing and plasmid profiling were used to assess the association between patient isolates and isolates from unused blood collection tubes. All E. americana isolates had similar antibiograms (i.e., resistance only to cephalothin) when tested at 37 degrees C. However, when the same isolates were tested for antimicrobial susceptibility at 25 degrees C, a different antibiogram (i.e., resistance to chloramphenicol, ampicillin, and cephalothin) was found. The majority of these isolates also demonstrated a unique four-plasmid profile (130, 56, 4.6, and 3.1 megadaltons), and two of these plasmids (130 and 56 megadaltons) were characterized as temperature-sensitive plasmids. An epidemiologic link between outbreak-associated isolates obtained from different time periods in the outbreak was supported by evidence of a significant trend in the ability of the outbreak-associated isolates to reduce nitrate, together with the presence of the resistance antibiogram at 25 degrees C and the demonstration of the unique four-plasmid profile.